The expression of VE-cadherin in breast cancer cells modulates cell dynamics as a function of tumor differentiation and promotes tumor–endothelial cell interactions

Abstract Purpose To examine the involvement of the F11R/JAM-A protein in breast cancer metastasis, we utilized the F11R/JAM-A antagonistic peptide 4D (P4D) in experiments of transendothelial migration (TEM) of breast cancer cells. Methods Experiments were conducted in the mouse 4T1 breast cancer model utilizing the human mammary epithelial cell and endothelial cell lines. The levels of soluble F11R/JAM-A (sJAM-A) in the murine plasmas were measured by ELISA. Levels of F11R/JAM-A mRNA and protein in cell lines were assessed by qRT-PCR and Western blot, respectively. Cell surface expression of F11R/JAM-A was demonstrated by flow cytometry. Functional tests included the TEM of breast cancer cells and adhesion of breast cancer cells to the endothelium. The endothelial permeability was studied by fluorescent tracer assay and by the Real-Time Cell Analysis (RTCA). Results The tumor inducers Tβ4 and TGF-β1 reduced the levels of sJAM-A in murine plasma, and reduced the F11R/JAM-A protein levels in the human microvascular endothelial cell line HMEC-1. The adhesion and TEM measured between breast cancer cells and inflamed or Tβ4-treated endothelium were inhibited by P4D. The presence of P4D did not destabilize the pre-existing tight junctions in the endothelial monolayer. The barrier-protecting effect of P4D was stronger than that of forskolin, when a booster dose of P4D was applied to the inflamed endothelium. Conclusions F11R/JAM-A protein can be considered as a novel target in the treatment of breast cancer metastasis. In vivo and clinical studies are needed to further investigate the effectiveness of F11R/JAM-A-derived peptide as a possible anti-metastatic drug.

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Liposomes Embedded in Layer by Layer Constructs as Simplistic Exosome Transfer Model

10.21203/rs.3.rs-46696/v1 ◽

2020 ◽

Author(s):

Gerardo Prieto ◽

Vicente Domínguez-Arca ◽

Rui R. Costa ◽

Ana M. Carvalho ◽

Pablo Taboada ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Nile Red ◽

Cellular Membrane ◽

Cell Interactions ◽

Bioactive Molecules ◽

Transfer Model ◽

Layer By Layer ◽

Dependent Manner

Abstract Background. Exosomes are extracellular vesicles originating from the exfoliation of the cellular membrane. They are involved in cell-to-cell and cell-to-matrix signaling, exchange of bioactive molecules, tumorigenesis and metastasis, among others. To mitigate the limited understanding of exosome transfer phenomena, we developed a simplistic model that mimics exosomes and their interactions with cells and the extracellular matrix. The proposed model is a layer-by-layer (LbL) film built from the polycationic poly-L-lysine (PLL) and the glycosaminoglycan hyaluronic acid (HA) to provide ECM mimicry. Positively charged 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and N1,N1,N14,N14-tetramethyl-N1,N14-ditetradecyltetradecane-1,14-diaminium dibromide (GS14) liposomes were embedded in this construct to act as exosome analogs.Results. To simulate exosomes carrying substances, Nile Red was loaded as a model of lipophilic cargo molecules. The integration of each component was followed by quartz-crystal microbalance measurements, which confirmed the immobilization of intact liposomes on the underlying (PLL/HA)3 soft film. The release of Nile Red from liposomes either embedded in the LbL construct or exposed at its surface revealed a fast first order release. This system was validated as a model for exosome/cell interactions by incubation with breast cancer cells MDA-MB-231. We observed higher internalization for embedded liposomes when compared with surface-exposed ones, showcasing that the ECM mimic layers do not constitute a barrier to liposome/cell interactions but favor them.Conclusions. Our findings indicate that the developed model enhances the structural stability of liposomes and induces endocytosis from breast cancer cells. We envisage that the internalization can be tuned by exploring different levels of embedment to achieve a cellular uptake modulated in a spatiotemporal dependent manner. The versatility provided by the LbL technique will allow incorporating additional specialized biomaterials to better mimic the structure and composition of exosomes and their role in cell-to-cell communications.

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